The STIM-Orai calcium signaling pathway plays a ubiquitous and central role in controlling growth, transcription, secretion, and development in many cell types including those of the immune system, muscle, nerve, skin, and blood. STIM proteins function with Orai channels to generate store-operated Ca2+ entry (SOCE), a fundamental signaling mechanism crucial to control of most cell types. Genetic defects in the STIM and Orai proteins are manifested prominently in many immune cells. Our specific aims are to understand the dynamic molecular coupling mechanism that occurs between the STIM Ca2+ sensor proteins in the endoplasmic reticulum and Orai Ca2+ channels in the plasma membrane, a mechanism crucial to mediating the generation of Ca2+ entry signals in all cells. Our studies utilize a number of important new molecular probes we have developed, and rest upon innovative mechanistic understanding we have recently presented on the ?coupling interface? between STIM and Orai proteins. This work has focused on (a) the molecular functioning of the active site on STIM proteins, (b) the intrinsic activation mechanism of Orai channels. Using model cell systems including B cells in which we can study and manipulate the machinery mediating SOCE, our specific aims are: 1: To determine the molecular basis of the STIM protein coupling interface: Our goals are to understand the molecular mechanism by which STIM1 and STIM2 and a novel splice variant, STIM2.1, become activated and interact with Orai channels to generate Ca2+ signals. Using new STIM-derived fluorescent molecular probes and a combination of high resolution FRET imaging and biophysical measurements, we challenge a current model involving unfolding of STIM proteins, and test a simpler gating interaction between STIM and Orai. 2: To determine the molecular basis of Orai channel function and gating by STIM proteins: Although the structure of Orai channels is now understood, the molecular arrangement of the channel and rearrangement that leads to channel opening with STIM is unknown. We have developed critical new tools with which to study activation and function of Orai channels. This includes a molecular modification of the Orai1 channel that mimics the STIM-activated open state of the channel which has never before been studied. Aim 3: To understand the organization and function of the Ca2+ entry coupling complex: The ER-PM junction wherein STIM and Orai proteins functionally interact has been the subject of much scrutiny but little understanding. Our studies are directed toward a hypothesis that STIM1 is able to cluster Orai1 channels within the ER-PM junction, and we examine how Ca2+ signals can be generated and how they activate the cellular transcriptional machinery in B cells. Through these aims, our studies provide important basic understanding of the crucial coupling interface between STIM and Orai proteins. Using B cell models the work has particular significance to understanding B cell function and development, providing important basic information on mechanisms to control major immunological diseases including primary B cell deficiencies, lymphoproliferative disorders such as chronic lymphocytic leukemia, and autoimmune diseases.